Regulatory T (Treg) cells are characterized by expression of both CD4 and CD25 and the forkhead/winged transcription factor FoxP3. First characterized in mice, in which they constitute about 6-10% of lymph node and splenic CD4+ T cell populations, CD4+CD25+ cells represent about 5-10% of human CD4+ T cells (Wing and Sakaguchi, 2010). As discussed in more detail below, Treg cells have the ability to suppress the activity of CD4+ T cells and CD8+ T cells.
Treg cells can be divided into several subsets (Bluestone et al., 2000). One subset of Treg cells develops in the thymus (also known as natural Treg (nTreg) cells), and these thymic-derived Treg cells function by a cytokine-independent mechanism, which involves cell to cell contact (Shevach, 2002). These cells are essential for the induction and maintenance of self-tolerance and for the prevention of autoimmunity (Shevach 2000; Salomon et al, 2000; Sakaguchi et al, 2001). These regulatory cells prevent the activation and proliferation of autoreactive T cells that have escaped thymic deletion or recognize extrathymic antigens, and, as a consequence are critical for homeostasis and immune regulation, as well as for protecting the host against the development of autoimmunity (Suri-Payer et al, 1996; Asano 1996; Willerford et al, 1995; Salomon et al, 2000).
Treg cells can also be generated by the activation of mature, peripheral CD4+ T cells (these cells are known as induced Treg (iTreg) cells). These cells can be generated ex vivo, e.g., by exposure to growth factors, and in vivo, e.g., in the gastrointestinal tract. Studies have indicated that peripherally derived Treg cells mediate their inhibitory activities by producing immunosuppressive cytokines, such as transforming growth factor-beta (TGF-β) and IL-10 (Kingsley 2002; Nakamura 2001). After antigen-specific activation, these Treg cells can non-specifically suppress proliferation of either CD4+ or CD8+ T cells (Baecher-Allan, 2001). Studies have shown that CD4+CD25+ cells are able to inhibit anti-CD3 stimulation of T cells when co-cultured with autologous antigen presenting cells (APC) (e.g., Stephens, 2001; Taams, 2001).
While both nTreg cells and iTreg cells have regulatory activity, a recent study indicates that iTreg cells lose this activity in vivo in a study of graft-versus-host disease (GVHD; Koenecke et al., 2009). In contrast nTreg cells maintain their regulatory activity and prevented development of GVHD. Thus, it is desirable to be able to identify/isolate populations of nTreg cells.
The immunomodulatory activity of Treg cells can be contact dependent, or the Treg cells may kill CD4+ and CD8+ T cells in a perforin- or granzyme-dependent manner or by the secretion of immunosuppressive cytokines, e.g., IL-10 and/or TGF-β (as reviewed in Dejaco et al., 2005).
Treg Cells and Autoimmunity/Tolerance
Depletion of Treg cells from various mouse strains has been shown to lead to a variety of autoimmune diseases that are tissue specific, including thyroiditis, oophoritis, gastritis or inflammatory bowel disease (Asano, 1996; Sufi-Payer, 1998; and McHugh, 2002). Furthermore, human patients having a mutation in the FoxP3 gene fail to produce Treg cells and develop autoimmune polyendocrinopathy (especially type I diabetes and hypothyroidism) and enteropathy (summarized as immunodysregulation, polyendocrinopathy, enteropathy X-linked (IPEX) syndrome). A polymorphism in the FoxP3 is associated with autoimmune diabetes (Bassuny et al, 2003). Moreover, mice deficient in FoxP3 develop an IPEX like syndrome (see, Dejaco et al., 2005).
The level of Treg cells in the circulation is reduced in subjects suffering from a variety of disorders as shown in Table 1. Moreover, lower levels of these cells are associated with higher disease activity and/or poorer prognosis.
TABLE 1Autoimmune diseases associated with reduced levels of circulating CD4+CD25+ cells.Level of CD4+CD25+ cells (control) Diseaseand significanceReferenceJuvenile idiopathic1.2 (1.6) ***De Kleer arthritis(2004)Juvenileidiopathic0.4 (1.2) ***Cao et al., arthritis(2004).Rheumatoid arthritis0.7 (1.2)*Cao et al., supra.Rheumatoid arthritis1.2 (3.7)*Liu et al., (2004).Psoriatic arthritis0.6 (1.2)*Cao et al., supra.HCVmixed2.6 (7.9)**Boyer et al., cryoglobulinemia(2004)Autoimmuneliver2.5 (6.8)***Longhi et al., disease(2004).Systemiclupus1.8 (3.7)*Cao et al., supra.erythematodes(SLE)SLE0.9 (2.6)*Crispin et al., (2003).*, p <0.05;**, p < 0.01,***, p < 0.001.
Increased levels of CD4+CD25+ T cells are observed at sites of inflammation, e.g., in subjects suffering from juvenile idiopathic arthritis, rheumatoid arthritis, sponyloarthritis and infections (as reviewed in Dejaco et al., 2005). These cells are considered to modulate local immune responses, e.g., to prevent collateral tissue damage.
Adoptive transfer of CD4+CD25+ T cells prevents the development of these diseases and, in some models can cure the disease after initiation (Mottet et al., 2003). Suri-Payer et al., (1998) also found that CD4+CD25+ T cells could prevent autoimmunity induced by autoantigen-specific T cell clones. Transfer of CD4+CD25− T cells into nude mice also leads to development of autoimmune disease, which can be prevented by co-transfer of CD4+CD25+ T cells (Sakaguchi et al., 1995).
Tang et al., (2004) demonstrated that Treg cells are also useful for the treatment of autoimmune diabetes. The authors isolated Treg cells from non-obese diabetic (NOD) mice and expanded TCR transgenic cells specific for an autoantigen. Adoptive transfer of these cells to NOD mice reversed diabetes in newly transgenic mice.
Studies by Trenado (2002) also demonstrated that infusion of ex vivo activated and expanded CD4+CD25+ T cells significantly inhibit rapidly-lethal graft-versus-host disease (GVHD) in mice. Treg cells have also been shown to suppress allograft rejection in rodents with long term surviving cardiac (Van Maurik, 2002) or pancreatic islet (Gregori, 2001) allografts.
Based on the foregoing, it will be apparent to the skilled artisan that Treg cells are attractive for treatment or prevention of autoimmune disease or for inducing tolerance in a subject or that the detection of circulating levels of Treg cells is useful for the diagnosis or prognosis of those disorders. However, difficulties have arisen in translating the results of animal models to the human situation as a result of insufficient markers that permit isolation of Treg cells. To date, the only marker that clearly distinguishes Treg cells from other T cells is FoxP3. FoxP3 is an intracellular protein and, as a consequence, is not useful as a marker for isolating viable Treg cells. Accordingly, there is a need in the art for new markers, preferably cell surface markers of Treg cells that permit detection and/or isolation of those cells, e.g., for diagnostic and/or therapeutic and/or prophylactic purposes.
Treg Cells and Inducing an Immune Response
Treg cells also exist in markedly higher proportions within tumor-infiltrating lymphocytes, peripheral blood lymphocytes, and/or regional lymph node lymphocytes of patients with cancer. The frequency of cells is related to tumor progression and inversely correlated with the efficacy of treatment. Accordingly, the ability of Treg cells to suppress immune responses appears to suppress the ability of the immune system to kill tumor cells.
Wang et al., (2004) isolated a CD4+CD25+ tumor-infiltrating lymphocyte (TIL) from a human melanoma patient. This TIL recognized a tumor/self-antigen, LAGE-1. CD4+CD25+ TILs have also been isolated from ascites of patients with ovarian cancer, and these cells were shown to be capable of suppressing T cell activity (Curiel et al., 2004).
Depleting populations of Treg has also been demonstrated to improve significantly the clearance of injected tumor cells. For example, Jones et al., 2002, depletion of CD25 expressing T cells using monoclonal antibody therapy facilitated long-term CD4+ T cell-mediated immunity against melanoma cells. The authors demonstrated that following anti-CD25 treatment, mice developed an immune response against a self-antigen (tyrosinase) that accompanies inhibition of tumor growth in mice.
Goforth et al., (2008) also demonstrated that poly lactic-co-glycolic acid (PLGA) polymer particles loaded with antigenic tumor lysate and immune stimulatory CpG oligonucleotides efficiently activated antigen-presenting cells and were incorporated into lysosomal compartments of macrophages and dendritic cells. Vaccination with the immune stimulatory antigen loaded particles (ISAPs) resulted in remarkable T cell proliferation, but only modestly suppressed tumor growth of established melanoma. When CD25′ cells were suppressed with anti-CD25 antibody, ISAP vaccination induced complete antigen-specific immunity in a prophylactic model. These findings suggest that it may be necessary or desirable to suppress Treg cell activity prior to and/or during vaccination, particularly against self-antigens.
It will be apparent from the foregoing discussion that depletion of Treg cells provides an attractive means for improving an immune response, e.g., against a self antigen or a non-self antigen. However, as discussed above, insufficient markers that permit removal of Treg cells has hampered therapeutic strategies targeting these cells. For example, while the cell surface marker CD25 is highly expressed on Treg cells and has been traditionally used to isolate or target these cells, this protein is also expressed on other T cell populations (e.g., activated T cells) in addition to activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes (see, for example, Robb et al., 1981; and Zola et al., 1989). Accordingly, there is a need in the art for new markers, preferably cell surface markers of Treg cells that permit detection and isolation of Treg cells, or removal or destruction of Treg cells, e.g., for diagnostic, prognostic, therapeutic and/or prophylactic purposes.